YBBRC 32797
No. of Pages 6, Model 5G
6 October 2014 Biochemical and Biophysical Research Communications xxx (2014) xxx–xxx 1
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc 5 6
GDC-0980-induced apoptosis is enhanced by autophagy inhibition in human pancreatic cancer cells
3 4 7 8 9 10 11 12 13 14 1 6 6 2 17 18 19 20 21 22 23 24 25
Q1
Jian-ying Tang a,1, Dai Tu b,1, Hui Zhang c, Wu-jun Xiong c, Ming-zheng Xu a, Xu-jing Wang c, Qing-he Tang c, Bo Chen c,⇑, Ming Xu d,⇑ a
Department of Emergency, East Hospital Affiliated to Tongji University in Shanghai, Shanghai, China Department of Hepatobiliary Surgery, Wuxi No. 2 People’s Hospital, Wuxi, Jiangsu Province, China Department of Biliary and Pancreatic Surgery, East Hospital Affiliated to Tongji University in Shanghai, Shanghai, China d Department of Gastroenterology, East Hospital Affiliated to Tongji University in Shanghai, Shanghai, China b c
a r t i c l e
i n f o
Article history: Received 22 September 2014 Available online xxxx Keywords: Pancreatic cancer PI3K–AKT–mTOR GDC-0980 Autophagy and chemo-sensitization
a b s t r a c t Pancreatic cancer remains fatal to the fast majority of affected patients. Activation of phosphoinositide-3 kinase (PI3K)–AKT–mammalian target of rapamycin (mTOR) pathway plays an important role in pancreatic cancer progression and chemo-resistance. In the present study, we examined the activity of GDC0980, a novel class I PI3K/mTOR kinase inhibitor, against pancreatic cancer cells in vitro. GDC-0980 inhibited AKT-mTOR activation and pancreatic cancer cell (PANC-1 and Capan-1 lines) survival. In both cancer cell lines, GDC-0980 simultaneously activated apoptosis and autophagy, the latter was detected by p62 degradation, Beclin-1 upregulation and light chain 3B (LC3B) conversion from a cytosolic (LC3B-I) to a membrane-bound (LC3B-II) form. Autophagy inhibitors including 3-methyladenine, hydroxychloroquine, NH4Cl and bafilomycin A1 enhanced apoptosis and cytotoxicity by GDC-0980, such an effect was reversed by caspase inhibitors (z-VAD-FMK and z-ITED-FMK). Furthermore, knockdown of LC3B or Beclin-1 through siRNA increased GDC-0980-induced anti-pancreatic cancer cell activity. Thus, inhibition of autophagy sensitizes GDC-0980-induced anti-pancreatic cancer activity, suggesting a novel therapeutic strategy for GDC-0980 sensitization. Ó 2014 Published by Elsevier Inc.
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44 45
1. Introduction
46
Pancreatic cancer remains fatal to the fast majority of affected patients, these patients were mostly diagnosed at the advanced stages [1–3]. The current standard treatments for pancreatic cancer include surgery (only for early stages), radiation and/or gemcitabine-based chemotherapies. However, pancreatic cancer is among the most intrinsically resistant malignancies to both radiation and chemotherapy, resulting in a median overall survival (OS) less than 7%. Hence, scientists including ours [4,5] and oncologists are searching for novel and more efficient anti-pancreatic cancer agents or adjuvants [1,6].
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Abbreviations: 3-MA, 3-methyladenine; BafA1, bafilomycin A1; Cq, hydroxychloroquine; LC3B, light chain 3B; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide-3 kinase; SE, standard error. ⇑ Corresponding authors at: No. 150, Ji-mo Road, Pudong New District, Shanghai 200120, China. Fax: +86 21 38804528 (B. Chen), +86 21 38804519 (M. Xu). E-mail addresses: [email protected] (B. Chen), [email protected] (M. Xu). 1 Co-first authors.
Phosphoinositide-3 kinase (PI3K)–AKT–mTOR pathway plays a vital role in cancer cell growth, survival and metastases [7–10]. In pancreatic cancer, this pathway could be activated by over-expression of growth factor receptors, as well as by mutation, amplification, and deletion of genes encoding components of the pathway [11–14]. For example, most pancreatic cancers have activating mutations in KRAS (Kirsten rat sarcoma viral oncogene homolog), resulting in an abnormal RAS protein that is ‘locked’ in the activated form [12,15,16]. This leads to aberrant activation of the RAS-mediated downstream signalings (i.e. PI3K–AKT–mTOR and Erk–MAPK) to promote cancer cell progression [12,15,16]. Activation of PI3K– AKT–mTOR pathway also mediates acquired resistance to both chemotherapies and molecule targeted agents [7–10]. Thus, therapeutic targeting of the PI3K–AKT–mTOR pathway with small molecule inhibitors may have clinical benefit for pancreatic cancer, either as single agents or used more broadly in combination with other conventional or targeted therapies. As a matter of fact, several inhibitors of this kind have now entered clinical trials [17,18]. GDC-0980 is a novel class I PI3K/mTOR kinase inhibitor. However, it activity in pancreatic cancer is somehow not as effective
http://dx.doi.org/10.1016/j.bbrc.2014.09.115 0006-291X/Ó 2014 Published by Elsevier Inc.
Please cite this article in press as: J.-y. Tang et al., GDC-0980-induced apoptosis is enhanced by autophagy inhibition in human pancreatic cancer cells, Biochem. Biophys. Res. Commun. (2014), http://dx.doi.org/10.1016/j.bbrc.2014.09.115
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YBBRC 32797
No. of Pages 6, Model 5G
6 October 2014 2
J.-y. Tang et al. / Biochemical and Biophysical Research Communications xxx (2014) xxx–xxx
cells (cytotoxicity) was calculated by the number of the trypan blue stained cells divided by the total number of the cells.
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79
as in other cancers [19]. Here we report that GDC-0980 activates autophagy in human pancreatic cancer cells. Inhibition of autophagy by pharmacologic or genetic means drives pancreatic cancer cells into apoptosis, and significantly enhances GDC-0980 activity.
2.7. Beclin-1 shRNA
130
80
2. Materials and methods
131
81
2.1. Cells
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Human pancreatic cancer cell lines (PANC-1 and Capan-1) were maintained in RPMI medium (Invitrogen, Shanghai, China), supplemented with 10% fetal bovine serum (FBS, Invitrogen), penicillin/ streptomycin (1:100, Invitrogen) and 4 mM L-glutamine (Sigma– Aldrich, St. Louis, MO) in a CO2 incubator at 37 °C.
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2.2. Reagents and chemicals
The targeted shRNA sequences for two different sites of human Beclin-1 were 50 -CTCAGGAGAGGAGCCATTT-30 [termed as shBeclin-1(-1)] [20] and 50 -CAGTTTGGCACAATCAATA-30 [termed as shBeclin-1(-2)] [21]. The annealed inserts were cloned into the RNAi-Ready pSIREN-RetroQ vector (Clontech) that had been previously digested with EcoRI-BamHI. The negative control shRNA (shSC) was purchased from Kaiji Biotech (Shanghai, China). The Beclin-1 shRNA (1/2) or a negative control shRNA was transfected into pancreatic cancer cells twice separated by 24 h using Lipofectamine and Plus transfection reagents (Invitrogen) according the protocol. The shRNA efficiency was verified by Western blotting. 2.8. LC3B siRNA
143
LC3B siRNA-1 (CS-6212, cellular signaling), LC3B siRNA-2 (sc43391, Santa Cruz Biotechnology) and LC3B siRNA-3 (LC-201, Kaiji Biotech, Shanghai, China) were purchased from commercial sources. Cells were cultured in six-well plates and transfected at 60% confluence. Transient transfection with 40 nM siRNA was performed with Lipofectamine and Plus reagents (Invitrogen), after 3 h, 1% FBS was added, and cells were left for another 36 h before they were trypsinized and used for experiments. The siRNA efficiency was again verified by Western blotting.
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GDC-0980 was obtained from Selleck China (Shanghai, China). Bafilomycin A1 (BafA1), 3-methyaldenine (3-MA), hydroxychloroquine (Cq), NH4Cl, and mouse monoclonal antibody against b-actin were obtained from Sigma–Aldrich Co. (St. Louis, MO). The broad caspase inhibitor z-VAD-fmk and the caspase-8 specific inhibitor z-ITED-fmk were from Calbiochem (Darmstadt, Germany). Antilight chain 3B (LC3B), Beclin-1 and rabbit/mouse IgG-horseradish peroxidase (HRP) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). All other antibodies were from Cell Signaling Tech (Denver, MA).
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2.3. Cell viability assay
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2.9. Statistical analyses
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Data were analyzed with SPSS 13.0 software using one-way analyses of variance (ANOVA). Differences between the control and treated samples were analyzed by using individual contrasts when the factor consisted of more than two levels. P values of
No. of Pages 6, Model 5G
6 October 2014 Biochemical and Biophysical Research Communications xxx (2014) xxx–xxx 1
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc 5 6
GDC-0980-induced apoptosis is enhanced by autophagy inhibition in human pancreatic cancer cells
3 4 7 8 9 10 11 12 13 14 1 6 6 2 17 18 19 20 21 22 23 24 25
Q1
Jian-ying Tang a,1, Dai Tu b,1, Hui Zhang c, Wu-jun Xiong c, Ming-zheng Xu a, Xu-jing Wang c, Qing-he Tang c, Bo Chen c,⇑, Ming Xu d,⇑ a
Department of Emergency, East Hospital Affiliated to Tongji University in Shanghai, Shanghai, China Department of Hepatobiliary Surgery, Wuxi No. 2 People’s Hospital, Wuxi, Jiangsu Province, China Department of Biliary and Pancreatic Surgery, East Hospital Affiliated to Tongji University in Shanghai, Shanghai, China d Department of Gastroenterology, East Hospital Affiliated to Tongji University in Shanghai, Shanghai, China b c
a r t i c l e
i n f o
Article history: Received 22 September 2014 Available online xxxx Keywords: Pancreatic cancer PI3K–AKT–mTOR GDC-0980 Autophagy and chemo-sensitization
a b s t r a c t Pancreatic cancer remains fatal to the fast majority of affected patients. Activation of phosphoinositide-3 kinase (PI3K)–AKT–mammalian target of rapamycin (mTOR) pathway plays an important role in pancreatic cancer progression and chemo-resistance. In the present study, we examined the activity of GDC0980, a novel class I PI3K/mTOR kinase inhibitor, against pancreatic cancer cells in vitro. GDC-0980 inhibited AKT-mTOR activation and pancreatic cancer cell (PANC-1 and Capan-1 lines) survival. In both cancer cell lines, GDC-0980 simultaneously activated apoptosis and autophagy, the latter was detected by p62 degradation, Beclin-1 upregulation and light chain 3B (LC3B) conversion from a cytosolic (LC3B-I) to a membrane-bound (LC3B-II) form. Autophagy inhibitors including 3-methyladenine, hydroxychloroquine, NH4Cl and bafilomycin A1 enhanced apoptosis and cytotoxicity by GDC-0980, such an effect was reversed by caspase inhibitors (z-VAD-FMK and z-ITED-FMK). Furthermore, knockdown of LC3B or Beclin-1 through siRNA increased GDC-0980-induced anti-pancreatic cancer cell activity. Thus, inhibition of autophagy sensitizes GDC-0980-induced anti-pancreatic cancer activity, suggesting a novel therapeutic strategy for GDC-0980 sensitization. Ó 2014 Published by Elsevier Inc.
27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43
44 45
1. Introduction
46
Pancreatic cancer remains fatal to the fast majority of affected patients, these patients were mostly diagnosed at the advanced stages [1–3]. The current standard treatments for pancreatic cancer include surgery (only for early stages), radiation and/or gemcitabine-based chemotherapies. However, pancreatic cancer is among the most intrinsically resistant malignancies to both radiation and chemotherapy, resulting in a median overall survival (OS) less than 7%. Hence, scientists including ours [4,5] and oncologists are searching for novel and more efficient anti-pancreatic cancer agents or adjuvants [1,6].
47 48 49 50 51 52 53 54 55
Abbreviations: 3-MA, 3-methyladenine; BafA1, bafilomycin A1; Cq, hydroxychloroquine; LC3B, light chain 3B; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide-3 kinase; SE, standard error. ⇑ Corresponding authors at: No. 150, Ji-mo Road, Pudong New District, Shanghai 200120, China. Fax: +86 21 38804528 (B. Chen), +86 21 38804519 (M. Xu). E-mail addresses: [email protected] (B. Chen), [email protected] (M. Xu). 1 Co-first authors.
Phosphoinositide-3 kinase (PI3K)–AKT–mTOR pathway plays a vital role in cancer cell growth, survival and metastases [7–10]. In pancreatic cancer, this pathway could be activated by over-expression of growth factor receptors, as well as by mutation, amplification, and deletion of genes encoding components of the pathway [11–14]. For example, most pancreatic cancers have activating mutations in KRAS (Kirsten rat sarcoma viral oncogene homolog), resulting in an abnormal RAS protein that is ‘locked’ in the activated form [12,15,16]. This leads to aberrant activation of the RAS-mediated downstream signalings (i.e. PI3K–AKT–mTOR and Erk–MAPK) to promote cancer cell progression [12,15,16]. Activation of PI3K– AKT–mTOR pathway also mediates acquired resistance to both chemotherapies and molecule targeted agents [7–10]. Thus, therapeutic targeting of the PI3K–AKT–mTOR pathway with small molecule inhibitors may have clinical benefit for pancreatic cancer, either as single agents or used more broadly in combination with other conventional or targeted therapies. As a matter of fact, several inhibitors of this kind have now entered clinical trials [17,18]. GDC-0980 is a novel class I PI3K/mTOR kinase inhibitor. However, it activity in pancreatic cancer is somehow not as effective
http://dx.doi.org/10.1016/j.bbrc.2014.09.115 0006-291X/Ó 2014 Published by Elsevier Inc.
Please cite this article in press as: J.-y. Tang et al., GDC-0980-induced apoptosis is enhanced by autophagy inhibition in human pancreatic cancer cells, Biochem. Biophys. Res. Commun. (2014), http://dx.doi.org/10.1016/j.bbrc.2014.09.115
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YBBRC 32797
No. of Pages 6, Model 5G
6 October 2014 2
J.-y. Tang et al. / Biochemical and Biophysical Research Communications xxx (2014) xxx–xxx
cells (cytotoxicity) was calculated by the number of the trypan blue stained cells divided by the total number of the cells.
128
79
as in other cancers [19]. Here we report that GDC-0980 activates autophagy in human pancreatic cancer cells. Inhibition of autophagy by pharmacologic or genetic means drives pancreatic cancer cells into apoptosis, and significantly enhances GDC-0980 activity.
2.7. Beclin-1 shRNA
130
80
2. Materials and methods
131
81
2.1. Cells
82
86
Human pancreatic cancer cell lines (PANC-1 and Capan-1) were maintained in RPMI medium (Invitrogen, Shanghai, China), supplemented with 10% fetal bovine serum (FBS, Invitrogen), penicillin/ streptomycin (1:100, Invitrogen) and 4 mM L-glutamine (Sigma– Aldrich, St. Louis, MO) in a CO2 incubator at 37 °C.
87
2.2. Reagents and chemicals
The targeted shRNA sequences for two different sites of human Beclin-1 were 50 -CTCAGGAGAGGAGCCATTT-30 [termed as shBeclin-1(-1)] [20] and 50 -CAGTTTGGCACAATCAATA-30 [termed as shBeclin-1(-2)] [21]. The annealed inserts were cloned into the RNAi-Ready pSIREN-RetroQ vector (Clontech) that had been previously digested with EcoRI-BamHI. The negative control shRNA (shSC) was purchased from Kaiji Biotech (Shanghai, China). The Beclin-1 shRNA (1/2) or a negative control shRNA was transfected into pancreatic cancer cells twice separated by 24 h using Lipofectamine and Plus transfection reagents (Invitrogen) according the protocol. The shRNA efficiency was verified by Western blotting. 2.8. LC3B siRNA
143
LC3B siRNA-1 (CS-6212, cellular signaling), LC3B siRNA-2 (sc43391, Santa Cruz Biotechnology) and LC3B siRNA-3 (LC-201, Kaiji Biotech, Shanghai, China) were purchased from commercial sources. Cells were cultured in six-well plates and transfected at 60% confluence. Transient transfection with 40 nM siRNA was performed with Lipofectamine and Plus reagents (Invitrogen), after 3 h, 1% FBS was added, and cells were left for another 36 h before they were trypsinized and used for experiments. The siRNA efficiency was again verified by Western blotting.
144
76 77 78
83 84 85
88 89 90 91 92 93 94 95 96 97
GDC-0980 was obtained from Selleck China (Shanghai, China). Bafilomycin A1 (BafA1), 3-methyaldenine (3-MA), hydroxychloroquine (Cq), NH4Cl, and mouse monoclonal antibody against b-actin were obtained from Sigma–Aldrich Co. (St. Louis, MO). The broad caspase inhibitor z-VAD-fmk and the caspase-8 specific inhibitor z-ITED-fmk were from Calbiochem (Darmstadt, Germany). Antilight chain 3B (LC3B), Beclin-1 and rabbit/mouse IgG-horseradish peroxidase (HRP) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). All other antibodies were from Cell Signaling Tech (Denver, MA).
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132 133 134 135 136 137 138 139 140 141 142
145 146 147 148 149 150 151 152
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2.3. Cell viability assay
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2.9. Statistical analyses
153
Data were analyzed with SPSS 13.0 software using one-way analyses of variance (ANOVA). Differences between the control and treated samples were analyzed by using individual contrasts when the factor consisted of more than two levels. P values of
